Project Summary The 3D topology of the genome plays a critical role in transcriptional regulation in health, disease and development. While techniques such as Hi-C and ChIA-PET have revealed static views of global genome architecture, very little is known about the mechanisms that control dynamic topological rearrangements. We have established a system in budding yeast ? the Hsf1-mediated heat shock response ? in which concerted structural rearrangements within and between a set of specific target genes take place. During heat shock, Hsf1 drives its target genes dispersed on different chromosomes to undergo striking changes in conformation and coalesce into discrete, transcriptionally active foci. These Hsf1 target genes encode a dedicated group of protein homeostasis (proteostasis) factors. Through their regulation, the human orthologue of Hsf1 (hHSF1) has been suggested to be a clinical target in malignant cancers and in neurodegenerative diseases. Thus, this proposal has dual significance: it will both elucidate mechanisms that control chromatin conformational dynamics and reveal how Hsf1 coordinates expression of the proteostasis machinery. The Hsf1-mediated heat shock response in budding yeast represents an ideal model system to investigate the regulation and function of 3D genome rearrangements for two reasons: 1) The magnitude, rapidity and specificity of the intra- and intergenic rearrangements are all unprecedented and thus represent exciting, novel biology; and 2) It will allow us to leverage the power of yeast gene- tics to dissect the mechanisms and define the functional relevance of genome topology dynamics. Aim 1 will focus deeply on the mechanisms that drive Hsf1 target gene interaction dynamics during heat shock. The experiments will utilize a modified version of chromosome conformation capture (3C) optimized to generate high-resolution interaction maps at targeted genomic loci in yeast coupled to molecular genetics to define the cis-acting DNA sequence elements and chromatin- associated proteins necessary for dynamic interactions within and between Hsf1 target genes. Aim 2 will investigate the 3D rearrangements that occur genome-wide during heat shock. The experiments will use Hi-C and ChIA-PET approaches and assess the functional role of Hsf1 and chromosomal topology and remodeling factors in orchestrating these changes. In support of the NIH mission, the precedents established in this proposal will inform therapeutic efforts aimed broadly at 3D genome regulation and may suggest novel molecular handles with which to modulate Hsf1 and the proteostasis machinery to treat cancer and neurodegenerative diseases.